Covalent modifications of DNA and RNA occur in every organism. They play important roles in determining the structure of nucleic acids, in gene regulation and in distinguishing native genetic material from the foreign. Of the base modifications found in nature, methylation of position 5 of cytosine is the most common. It is also known that 5-methyl-cytosine (5mC) is unstable and that it spontaneously deaminates to thymine. As a result, sites of cytosine methylation are found to be "hot-spots" for C to T transition mutations. We will investigate two aspects of cytosine methylation in the cells- the act of methylation itself and a mechanism built-in to defend the cells from the chemical consequences of methylation. In one part of the project we will study the EcoRII methylase for its catalytic mechanism and its base sequence-specific interaction with DNA. Recently, DNA cytosine methylases have been identified to have several conserved domains. We will test the hypothesis that while the conserved domains reflect the common catalytic mechanism for the transfer of methyl group to position 5 of cytosine, the unique segment(s) give the ability to recognize different base sequences to different enzymes. We will also study a DNA mismatch correction process in Escherichia coli called Very Short-patch (VSP) Repair which corrects T:G mismatches to C:G. The process is sensitive to the sequence surrounding the mismatch in such a way that whenever 5mC deaminates to T, the resulting mismatch is subject to correction. A similar process has recently been discovered in mammalian cells. We have identified a gene required for VSP repair. We will investigate the regulation of this gene and identify the biochemical role of its product in the repair process. These studies should enhance our understanding of DNA methylation and of its consequences to the cell and should provide insights in the interaction of enzymes with DNA.